RU2233670C2 - Cultural inactivated vaccine against chlamydiosis in pigs - Google Patents

Abstract

FIELD: veterinary microbiology, vaccines.

SUBSTANCE: vaccine comprises chicken embryo fibroblast suspension infected with chlamydia of the strain Chlamydia psittaci (var. suis) VGNKI "SR" № 14 as an antigen with titer value 10^-7 TCD_50/ml, 99.0-99.5; phenol, 0.3-0.5; polyvinylpyrrolidone, 0.4-0.6 mas. Vaccine elicits high immunogenicity and harmlessness.

EFFECT: enhanced effectiveness of vaccine.

6 tbl

Description

The invention relates to veterinary microbiology and virology, in particular to the production of vaccines used for specific prophylaxis and treatment of farm animals from chlamydia disease. The vaccine contains as an antigen a cytopathogenic strain of Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, grown on a monolayer of the primary culture of chicken embryo fibroblast cells (FEC) with a titer of at least 10 ^-7 TCD _{50 / ml} . The vaccine also contains an inactivator - phenol and the first used adjuvant - polyvinylpyrrolidone. The vaccine is specific, highly immunogenic and has a low reactivity.

The invention relates to veterinary microbiology and virology, in particular to the production of vaccines used for specific prophylaxis and therapy of farm animals from chlamydia disease.

Known vaccine against enzootic abortion of sheep inactivated, made from chlamydia-infected chicken embryos (Franc et al. Am. J. Vet. Pec., Vol. 29, p. 1441, 1968). The disadvantage of this vaccine is its high cost, its manufacture is time-consuming, the high content of ballast proteins reduces its immunogenicity and increases reactogenicity. This vaccine does not contain a specific antigen and therefore is not immunogenic for birds. We selected this vaccine as a prototype.

The closest in technical essence is US patent No. 4386065, A 61 K 39/118, 1983. "Vaccine against enzootic abortion of sheep", which includes a mixture of an aqueous suspension, formalin inactivated chlamydial elementary bodies obtained from chlamydia SP organisms ranging from 2 · 10 ⁶ to 10 ⁹ ELD _{50 / ml} , propagated in cell culture, and an equal amount of an oil adjuvant, which is a mixture of light mineral oil and lanolin.

The disadvantage of this vaccine is that the strain of chlamydia, as a producer, does not have a cytopathic effect. As a result, to control the accumulation of chlamydia, it is necessary to prepare smears daily, stain them and count infected cells, and when collecting raw materials, the infected monolayer must be separated from the growth surface using trypsin with versts. To create the desired concentration of chlamydia in the vaccine, the culture suspension is centrifuged. In addition, one stationary monolayer is used as seed for only five pure monolayers or their equivalents. All these operations complicate the manufacturing process and reduce productivity in the manufacture of the vaccine, which makes it more expensive. In addition, the vaccine prevents only enzootic chlamydial abortion in sheep and does not prevent chlamydia in pigs.

SUMMARY OF THE INVENTION

The objective of the invention is the creation of a specific, highly immunogenic vaccine for the prevention of chlamydia of pigs, which has low reactogenicity and is obtained more simply (in comparison with the prototype) technological method. This object was achieved due to the fact that the vaccine culture inactivated against chlamydia of pigs as an antigen contains the cytopathogenic strain of Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, grown on a monolayer of the primary culture of chicken embryonic fibroblast cells with a titer of at least 10 ^-7 TCD _{50 / ml} , for the first time we used polyvinylpyrrolidone as well as phenol in the following percent ratios:

1. Suspension of cells FEC (fibroblasts of chicken embryos) infected with chlamydia strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 with a titer of at least 10 ^-7.0 TCD _{50 / ml} - 99-99.5.

2. Phenol - 0.3-0.5.

3. Polyvinylpyrrolidone - 0.4-0.6.

A significant difference between this vaccine and its prototype is that the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, deposited in the VGNKI collection of veterinary preparations, is taken as antigen. Its cultivation is carried out on a monolayer of the primary culture of FEC.

The aim of the invention is to obtain a strain of Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, with stable immunogenic properties, low reactogenicity and high cytopathic effect. Vaccination with an inactivated vaccine made from Chlamydia psittaci (var. Suis) strain VGNKI "SR" No. 14 of pregnant sows when administered intramuscularly twice at a dose of 10 ml ensures the safety of newborn piglets by 93.3% versus 43.6% in unvaccinated control animals ( table 2).

The strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 is deposited in the collection of the All-Russian State Research Institute for Control, Certification and Standardization of Veterinary Medicines under number 14. The strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 is allocated in 1976 from an aborted sow fetus, adapted to a monolayer of chicken embryo fibroblasts (FEC). The strain was passivated through the pig’s body and passed 307 passages into the monolayer of the FEC culture under the conditions of a roller, as a result of which its initial properties changed: the accumulation ability, expressed in embryonic lethal doses (ELD _50/03 ml) in the indicated culture, increased, the development cycle of cytoplasmic inclusions accelerated In this connection, the ability of the strain to exert a high cytopathic effect on the monolayer of culture (FEC) appeared, along with this, immunogenic activity steadily increased and reactogenicity decreased.

The bacterial strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 has the following properties.

MORPHOLOGICAL SIGNS. Elementary mature particles are rounded in shape, with a diameter of 180-200 nm, initial intermediate forms - up to 500-600 nm, are stained pink-red according to the Stamp method, and according to Romanovsky-Giemsa they are dark blue.

CULTURAL PROPERTIES. It is cultivated in the vitelline membranes of 7-8-day-old chicken embryos, in the culture of chicken embryo fibroblasts, in the transplanted line of the calf’s trachea at 37-37.5 ° С; Passaged on white mice with intraperitoneal, nasal, intracerebral and subcutaneous infection.

ADAPTATION PROPERTIES. The strain is the causative agent of pig chlamydia, adapts to chicken embryos, while maintaining pathogenicity for pigs, adapts to chicken fibroblast cultures, increases virulence and immunogenicity, while the titer for titration on 6-7-day-old chicken embryos is 10 ^6.5 -10 ^7.5 ELD _50/03 ml and the development cycle time is reduced.

PATHOGENIC PROPERTIES. The strain is pathogenic for guinea pigs, causes abortion in them, the birth of dead cubs and the death of some infected animals, is pathogenic for white mice, causes disease and 100% death on days 4-6 with intracerebral administration.

ANTIGENIC PROPERTIES. In the formulation of the cross complement binding reaction with homologous and heterologous antigens and hyperimmune sera, a high antigenic relationship to the homologous antigen was established in a titer of 1: 40-1: 80 (100% hemolysis in the complement binding reaction (CSC)). Antigenic proximity to other types of chlamydia antigens is 3-4 dilutions further.

TAXONOMIC CHARACTERISTIC. According to the decision of the Legal Commission of the International Association of Microbiological Societies (MAMO) from January 1, 1980, the definition of the microorganisms in question, as Chlamydia, became mandatory. According to "The Shorter Bergey's manual of determinative" (1980), chlamydia is assigned to the kingdom of Procaryotae, a division of 2 prokaryotes indifferent to light, i.e. "cattle bacteria", class 2 Ricketsia. The Ricketsia class includes orders of 1 Ricketsialesu 2 Chamydiales. The family of Chlamydiaceae, the genus Chlamydia, which describes 4 species: Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pecorum, Chlamydia Pneumoniae. All types of chlamydia are heterogeneous.

IMMUNOGENOUS PROPERTIES. Example 1. In the experiment, 24 white mice were vaccinated with an inactivated vaccine made from strain "SR". The vaccine is administered subcutaneously at a dose of 0.2 ml.

The titrated control strains “P” at a dose of 0.3 ml with a titer of 10 ^6.0 LD _{50 / 0.3} ml 14 days after the first injection of the vaccine, 12 experimental and 12 control white mice were infected subcutaneously. Twenty days after vaccine administration, 12 vaccinated and 12 unvaccinated white mice are also infected. The results are presented in table. 1, where it is shown that the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 has immunogenic properties, which allows it to be used for the manufacture of an inactivated vaccine against chlamydia of pigs.

Example 2. The prepared inactivated vaccine against chlamydia of pigs from strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 is tested in experiments on pigs. The vaccine is administered intramuscularly, twice with an interval of 6 days, at a dose of 10 ml of six clinically healthy pregnant sows. As a control, 6 pregnant unvaccinated sows are used. Vaccinated and control animals after 24 days are infected intraperitoneally with a suspension of chlamydia isolated from the parenchymal organs of a stillborn piglet in a titer of 10 ⁷ ELD ₅₀ and in a volume of 20 ml.

The results of the immunogenicity of the vaccine are presented in table. 2.

Thus, the vaccine made from the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 ensures the safety of piglets in vaccinated sows 93.3% against 43.6% in unvaccinated animals.

The second significant difference is that polyvinylpyrrolidone (PVP) is administered as an adjuvant to the vaccine. Polyvinylpyrrolidone - a white powder, highly soluble in water, is used as a drug prolongator. It has an inherent detoxification and anti-inflammatory effect. In our preparation, PVP significantly increases the immunogenicity of the vaccine due to the fact that it binds (adsorbs) antigenic proteins.

Comparative analysis with the prototype allows us to conclude that the claimed composition of the vaccine differs from the known introduction of new components, namely the new strain of Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 with a titer of at least 10 ^-7.0 TCD _{50 / ml} and polyvinylpyrrolidone as an adjuvant, as well as the ratio of components.

Thus, the claimed technical solution is new. The introduction of polyvinylpyrrolidone as an adjuvant significantly increases the immunogenicity of the vaccine compared to a vaccine containing paraffin oil as adjuvant. To prove the following experiment was carried out. Eight guinea pigs were given a culture inactivated vaccine made from Chlamydia psittaci (var. Suis) strain VGNKI SR No. 14 containing paraffin oil as an adjuvant, and eight guinea pigs were given a vaccine containing polyvinylpyrrolidone as an adjuvant. The vaccine was administered subcutaneously, three times with an interval of 7 days at a dose of 0.2 ml. The immunogenicity of the vaccine was evaluated by the results of CSC (with the blood serum of experimental guinea pigs taken 20 days after the last injection of the vaccine). The results are presented in table. 3.

From the table. 3 it follows that the immunogenicity of the vaccine with polyvinylpyrrolidone used as adjuvant is significantly higher than vaccines with paraffin oil used as adjuvant.

Thus, this composition of the components gives the vaccine new properties, which allows us to conclude that the claimed technical solution meets the criterion of "inventive step".

Example 1 (best option)

Preparation of a cell culture of FEC, infection with its chlamydia was carried out according to the generally accepted method (L.P. Dyakonov et al. Cultivation of animal cells and tissues. Part 1. - Stavropol, 1986). Then, a suspension of FEK cells infected with chlamydia of the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 was subjected to control seeding for bacterial and fungal contamination. After that, a clean, non-contaminated suspension was taken to determine the titer of TCD _{50 / ml} . The titer was determined according to the generally accepted method and was calculated according to the method of Reed and Mench. In this example, it is equal to 10 ^-7.0 TCD _{50 / ml} .

Then the components were mixed in the following proportions: suspension of cells of FEC infected with chlamydia, 99.1 wt.%, Phenol 0.35 wt.%, Polyvinylpyrrolidone 0.55 wt.%. The resulting mixture was kept for 96 hours at a temperature of + 10 ° C.

Then, selective immunogenicity control was performed. For this, eight guinea pigs were taken and a vaccine was administered to them according to the following scheme:

1st introduction - subcutaneous 0.2 ml;

2nd introduction - subcutaneously after 7 days, 0.2 ml;

3rd introduction - subcutaneously after 14 days with 0.2 ml.

The control was four guinea pigs to which the vaccine was not administered. Three weeks after the last injection of the vaccine, the level of accumulation of antibodies in the blood serum of guinea pigs in the complement binding reaction (CSC) was determined. Result: four crosses at a dilution of 1: 5, four at a dilution of 1:10, two at a dilution of 1:20.

Similarly done for a titer of 7.5; 7.75; 6.5; 6.0; 5.5 TCD _{50 / ml} Information is given in table. 3.

Thus, the titer of chlamydial strain 7.0 provides a high accumulation of antibodies in the blood serum of guinea pigs. The vaccine with the quality described in examples 1, 4, 5, is considered suitable for use in pigs. In order to save strain material when obtaining a titer above 7.8 TCD _{50 / ml, the} suspension is diluted with GLA nutrient medium to the titer shown in examples 1, 4, 5. The titer of the strain below 6.5 TCD _{50 / ml} (example 6) does not provide the necessary accumulation of chlamydia and is not used for immunization of guinea pigs.

In this experiment, the dependence of the titer of antibodies in the blood serum of guinea pigs on the titer of the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 is clearly visible. This test was taken by us as a control of the immunogenicity of the vaccine in its manufacture.

Example 7. The preparation of the cell culture FEC, infection with its chlamydia, growing and accumulating them was carried out according to the standard method. Then, a suspension of PEC cells infected with chlamydia strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, was subjected to control seeding to check for bacterial and fungal contamination and at the same time determined the average titer of TCD _{50 / ml} , which in this example is 10 ^{-7 , 2} TCD _{50 / ml} .

The pure, non-contaminated bacterial and fungal flora cell chlamydial suspension was combined into a common sterile container and chlamydia phenol was inactivated. To do this, the components were mixed in a container in the following ratio: 99.2 wt.%, Suspensions of FEC cells infected with chlamydia were taken, and 0.4 wt.% Was added to the inactivator (phenol). The resulting mixture was kept for 98 hours at a temperature of + 10 ° C. Then, 0.4 wt.% Adjuvant (polyvinylpyrrolidone) was added. Then, selective control was carried out for the completeness of inactivation. To this end, 0.3 ml of the vaccine was sterilely introduced into 10 developing chicken embryos of a 7-day incubation, and they were incubated at 37.0-37.5 ° C for 12 days. As a control, 10 embryos were used, which were injected with a non-inactivated cell suspension containing the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14. The incubation conditions are the same. The experimental embryos remained alive for 12 days. Chlamydia was not found in smear prints made from the shells of the yolk sac, stained by the Romanovsky-Giems method. Control embryos fell on the 5-7th day. This result corresponds to the established control of the vaccine for completeness of inactivation.

Similarly, they were made for the following amounts of phenol: 0.3 wt.%; 0.2 wt.%; 0.5 wt.%; 0.6 wt.%. The results are shown in table. 4. Based on the data given in table. 4, it is seen that the amount of inactivator in the range from 0.3 to 0.5 wt.% Ensures the complete inactivation of chlamydia in suspension. Above 0.5 wt.% Phenol is not added in order to save it, because it does not improve the quality of the vaccine. The inactivator concentration below 0.3 wt.% Is not allowed, because this does not ensure complete inactivation of chlamydia.

Example 12. Preparation of the FEC culture, infection with its chlamydia, their cultivation and accumulation was carried out according to the generally accepted method. Then, a suspension of PEC cells infected with chlamydia of the Chlamydia psittaci strain (var. Suis) of VGNKI "SR" No. 14 was subjected to control seeding to check for bacterial and fungal contamination. If the test turned out to be negative, then the titer of TCD _{50 / ml was} selectively determined, which in this example is 7.3 TCD _{50 / ml} . Then the components were mixed in the following proportions: suspension of cells infected with chlamydia was taken 99.3 wt.%, Phenol 0.3 wt.% (The mixture was kept for 10 ° hours at -6 ° C). Polyvinylpyrrolidone was administered 0.4 wt.%. The process was carried out as follows: a weighed adjuvant and an inactivated cell suspension containing the antigen of the strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14 were left at room temperature until it was completely dissolved - 24 hours. Then, the immunogenicity control was carried out in the same way as in Example 1. The result is as follows: at a dilution of 1: 5 - ++++; at a dilution of 1:10 - ++++; at a dilution of 1:20 - ++. Similarly done for 0.3 wt.%. The results are shown in table. 5.

As indicated in the table. 5, the adjuvant content in the range of values from 0.4 to 0.6 wt.% Ensures the immunogenicity of the vaccine in accordance with the established control. The amount of adjuvant in excess of 0.6 wt.% Is not used to save it. A significant improvement in the quality of the vaccine with an increase in its adjuvant content in excess of 0.6 wt.% Does not occur.

The adjuvant content below 0.4 wt.% Is not allowed, because the immunogenicity of such a vaccine is lower than the established control of the vaccine and it does not cause a reliable immune response in birds.

Example 17. The preparation of the cell culture FEC, infection with its chlamydia, their growth and accumulation was carried out according to the standard method. Then, a suspension of FEK cells infected with the Chlamydia psittaci strain (var. Suis) of VGNKI "SR" No. 14 was subjected to control seeding to check for bacterial and fungal contamination. With a negative test result, the average titer of TCD _{50 / ml was} determined, which in this example is 7.4 TCD _{50 / ml} . Then, the components were mixed in the following proportions: suspensions of FEC cells infected with chlamydia were taken 99.25 wt.%; phenol - 0.35 wt.%, polyvinylpyrrolidone - 0.4 wt.%. Then, immunogenicity control was carried out, as in Example 1. The results of CSC are as follows: at a dilution of 1: 5 - ++++; at a dilution of 1:10 - ++++; at a 1:20 dilution - +++. Similarly done for 99; 98; 99.5 and 99.7 wt.% Suspension of FEC cells infected with a strain of Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14. The result is presented in table. 6.

As indicated in the table. 6, the content of the suspension of FEC cells infected with chlamydia strain Chlamydia psittaci (var. Suis) VGNKI "SR" No. 14, in the range from 99.0 to 99.5 wt.% Provides the immunogenicity of the vaccine in accordance with its control. The amount of cell culture suspension above 99.5 wt.% Is not used, because this does not improve the quality of the vaccine, but increases the consumption of its expensive components. The use of cell culture suspension in an amount below 99.0 wt.% Is also not allowed, because the vaccine does not have immunogenicity corresponding to the established control of the vaccine, and does not cause the development of reliable immunity in pigs.

TECHNICAL RESULT

The vaccine prevents the occurrence of chlamydia on the basis of sows: chronic infertility (lengthening the service period and arrhythmias of the reproductive cycle), miscarriages, abortions, the birth of a dead offspring, the birth of clinically ill and latently infected piglets, and chronic pneumonia.

In piglets: enteritis, bursitis, bronchopneumonia, growth retardation and development on the background of immunodeficiency.

In boars: chronic pneumonia, arthritis, bursitis, inflammation of the spermatic cord.

In addition, the vaccine reduces environmental pollution with chlamydia, which are anthropozoonoses, and also reduces the costs of a dysfunctional farm for conducting ineffective treatment and prophylactic measures using antibiotics and other therapeutic agents. The vaccine is harmless. In general, the effectiveness of the vaccine is 90-93%.

Sources of information

1. L.P. Dyakonov, V.F. Glukhov and others. Cultivation of animal cells and tissues. Educational-methodical manual (part 1). - Stavropol, 1986

2. Description of US patent No. 4386065, MKI A 61 K 39 /, 1983.

3. A.S. N 645369, s. N 2528058, MKI C 12 K 5/00.

4. A.S. N 660390, z. N 2528670, MKI C 12 K 5/00.

5. A.S. N 661017, z. N 2528672, MKI C 12 K 5/00.

6. A.S. N 666203, z. N 2528672, MKI C 12 K 5/00.

7. A.S. N 666202, z. N 2528669, MKI C 12 K 5/00.

8. A.S. N 655160, s.N 2528668, MKI C 12 K 5/00.

9. Description of the patent No. 2124465.

Claims (1)

Pig chlamydia vaccine is a culture inactivated vaccine containing a chlamydial antigen, adjuvant and inactivator, characterized in that it contains a suspension of chicken embryo fibroblast cells (FEK) infected with chlamydia strain Chlamydia psittaci (var. Suis) VGNKI SP No. 14 titer no lower than 10 ^-7 TCD _{50 / ml} , inactivator - phenol and adjuvant - polyvinylpyrrolidone in the following ratio of ingredients, wt.%: Suspension of FEK cells infected chlamydia strain Chlamydia psittaci (var. suis) VGNKI "SR" No. 14 with a titer of at least 10 ^-7 TCD _{50 / ml} 99.0-99.5 Phenol 0.3-0.5 Polyvinylpyrrolidone 0.4-0.6